1. Field of the Invention
The present invention is broadly concerned with methods of inhibiting leukocyte superoxide anion (O2xe2x88x92) production and methods of attracting a leukocyte to a location using peptides. More particularly, the invention relates to methods employing a naturally occurring proline-arginine (PR)-rich antimicrobial peptide known as PR-39 and analogs thereof; these peptides can be used as medicaments that fight infection by attracting leukocytes to a wound site, yet restrict tissue damage at the wound site caused by excessive oxygen radicals produced by these leukocytes.
2. Description of the Prior Art
Infectious diseases are a primary cause of morbidity and mortality in humans and animals. These maladies range from the troublesome, e.g., Escherichia coli diarrhea which is caused by the consumption of contaminated food and drinks, to the deadly, e.g., AIDS.
A variety of antimicrobial agents have been developed to combat infectious diseases. Recently, several types of antimicrobial peptides have been discovered. Such peptide antimicrobials are produced by many biological organisms and are important components of host defense mechanisms. (Boman, 1991; Zasloff, 1992; Gabay, 1994; Boman, 1995, Martin et al., 1995) For example, defensins are expressed in several mammalian species (Lehrer et al., 1993), magainins have been identified in the skin and intestine of frogs (Zasloff, 1987; Moore et al., 1992), and cecropins have been isolated from insects and pigs (Steiner et al., 1981; Lee et al., 1989). These natural antimicrobials are lytic peptides that kill microorganisms by pore-forming, membrane-damaging mechanisms (Boman et al., 1993; Maloy et al., 1995).
The patent art also discloses antimicrobial peptides. U.S. Pat. No. 5,234,716 describes broad spectrum tryptophan antimicrobial peptides, and U.S. Pat. No. 5,202,420 discloses tracheal antimicrobial peptides.
Recently, a group of PR-rich antibacterial peptides have been identified. Bactenecins 5 and 7 have been isolated from bovine neutrophils (Gennaro et al., 1989; Litteri et al., 1993), and PR-39 was first isolated from the porcine small intestine (Agerberth et al., 1991) and identified recently in porcine and human neutrophils (Shi et al., 1994b; Shi et al., 1995). Although these PR-rich antibacterial peptides share a similar high content of proline (47, 47, and 49%, respectively) and arginine (21, 29, and 26%, respectively), they possess different killing mechanisms.
Similar to other lytic peptides, bactenecins kill bacteria by a membrane-permeability-associated mechanism (Maloy et al., 1995); however, PR-39 was found to kill bacteria by interfering with DNA and/or protein synthesis (Boman et al., 1993). Furthermore, PR-39 has been isolated from wound fluid and was shown to induce syndecan expression on mesenchymal cells (Gallo, 1994). Because syndecans are important in wound repair, this finding suggests that PR-39, in addition to its antibacterial properties, may have a larger role in inflammatory processes and tissue repair.
Neutrophils represent a first line of defense against infections; they are the first white blood cells to arrive at sites of infection and are well-equipped to sequester and eliminate pathogens. These cells possess multiple antimicrobial defense mechanisms, including both oxidative and nonoxidative microbial killing processes (Klebanoff, 1992; Selsted et al., 1995). Nonoxidative neutrophil defense mechanisms include several antibacterial peptides including PR-39. Phagocyte oxidative defense mechanisms are initiated by a plasma membrane-bound enzyme complex called reduced nicotinamide dinucleotide phosphate (NADPH) oxidase (Rotrosen, 1992). This multicomponent enzyme catalyzes the reduction of molecular oxygen to O2xe2x88x92 using NADPH as an electron donor. Although O2xe2x88x92 and other reactive oxygen intermediates are important components of host defense, these highly toxic oxidants also cause significant tissue injury in inflammatory diseases and ischemia-reperfusion injury (Shasby et al., 1982: Malech, 1987; Demling, 1990; Martinez-Cayuela, 1995; Granger et al., 1995). Thus, their generation and inactivation must be tightly regulated.
At least five proteins comprise the NADPH oxidase complex; a membrane flavocytochrome b558, which is composed of two subunits (gp91phox and p22phox); and three cytosolic components (p47phox, p67phox, and a GTP-binding protein named p21Rac) (Rotrosen et al., 1992; Abo et al., 1992). Although mechanisms for activation and assembly of NADPH oxidase have not been elucidated fully, it is clear that multiple protein-protein interactions among its components are regulated by a number of signaling intermediates (McPhail et al., 1993). The assembly of phagocyte NADPH oxidase requires protein-protein interactions between Src homology 3 (SH3) domains in cytosolic components and proline-rich regions in other components (Leto et al., 1994; Sumimoto et al., 1994; Finan et al. McPhail, 1994; de Mendez, 1996).
The present invention is predicated upon the discovery that specific peptides (e.g., PR-39) are capable of 1) inhibiting O2xe2x88x92 synthesis by leukocyte enzymes (e.g., NADPH oxidase), and 2) attracting leukocytes (e.g., neutrophils). These peptides can be used as novel medicaments that fight infection by attracting leukocytes to a wound site, yet restrict tissue damage at the wound site caused by excessive oxygen radicals produced by these leukocytes. Preferably, these peptides have a sequence included in PR-39 (e.g., Sequence ID Nos. 1 and 2 for peptides capable of inhibiting O2xe2x88x92 production, and Sequences ID Nos. 1, 2, 5, 6, and 7 for peptides capable of attracting leukocytes). Advantageously, these peptides are synthesized and have lengths of less than 60 amino acid residues, and the leucocytes upon which the peptides act are mammalian (e.g., porcine) leucocytes. This invention was made with government support under Grant 93-37206-9351 awarded by the United States Department of Agriculture. The government has certain rights in the invention.